In article <8c3faf6a.0110090414.598bb703 at posting.google.com> Nick
Jacobsen, nickjacobsen at yahoo.co.uk writes:
>Hi,
>>Can anyone help me with dissociated DRG cell culture. i prepare the
>culture and grow for 24hours in DMEM w/FCS/glutamine/NGF. On day 2 i
>change to neurobasal medium w/glutamine/B27/NGF and antimitotics FdU
>and uridine. After two days i change to neurobasal w/glu/B27/NGF. By
>day7 the cultures are dead and at this point id rather they were
>living,
>>can anyone help me out here ?
>>thanks very much in advance
>>Nick
Hi Nick,
Ahh, the old "dead neurons" problem.
There are so many possible causes of these problems that it's hard to
know where to start.
So first, some questions:
1) Are you starting up this culture method from scratch, or have the
cultures already been working well, and now are suddenly dying?
If the cultures were previously working, then the basic method &
solutions etc must be reasonably sound, and you should look for
-changes- in the procedure that caused the problem. some obvious
things that can change are:
a) the exact lot of serum you use. Different lots can be very
different in their composition, and dramatically alter your success.
If you are using a new or untested lot of serum, you should a) try and
get some of the old serum that was being used when things were still
working, b) literally test several different lots (the company will
usually provide small samples for this purpose, free of charge if you
ask). Make a few culture dishes and use a different lot on each group
of 2-3 dishes. -ALSO- test different percentages of serum, between
3-15%.
2) If you are not using an already established protocol (i.e., I mean
a protocol that someone else -in your lab- has already had success
with, not just one that has been published), then here are a few
suggestions:
a) Change the medium on day two, but don't use a new type of medium.
Just use the same medium you used for plating. I am not familiar with
B27, but I assume it's some sort of defined media additive or serum
substitute. Instead of this, try giving them the same serum they were
plated in. Neurons like serum, and robbing them of it when they are
still innocent babes can have dire consequences. After they've
established themselves, and look healthy, then you can try taking it
away and see what happens. If it is not absolutely essential to
interpreting your experiment that your cells be grown in completely
known media, then keep them in serum always. They will thank you for
it.
b) Neurons also like glia. Something that works very well is the
following: Plate cells during week#1. Allow them to grow up. DON'T add
FUdR until all the glial cells have reached confluency and covered the
whole dish. Then at the end of week#1, kill all the neurons by bathing
them in PBS + 100 micromolar glutamate for about 15 minutes. They
should all be dead the next day, but the glial cells won't. They'll
still be in a confluent monolayer covering the bottom of the dish.
Then, on week#2, plate your freshly dissociated cells on top of this
monolayer. After a few days, you can add antimitotics. Notice that
FUdR doesn't just halt mitosis, it -kills- mitotic cells. If you add
it while the glia are still proliferating, you will kill everything.
However, once the glia become confluent, they stop mitosis through
"contact inhibition". Then adding FUdR won't hurt the confluent cells
anymore, but it'll keep the newly plated glia from overgrowing
everything.
3) I cannot emphasize enough how critical the actual dissection and
dissociation proceedures are. You should dissect in ice cold PBS, and
the entire dissection shouldn't take more than 10 minutes. You should
slice up the tissue into tiny chunks mechanically, before putting it
the papain or trypsin. This exposes more surface area, and allows you
to use less enzyme and shorter incubations. You should triturate
-very- gently. If you have to squirt the cells back and forth more
than once or twice to dissociate them, that's probably damaging them
badly. Too much enzyme for too long will also damage them badly.
4) Finally, the final plating density can also be a critical factor.
Try plating 2-5 times as many cells and see if that improves survival.
If it does, it -does not- mean that the higher density is the right
one. But it tells you that something about density is important for
fixing the problem, and supports the idea that the cells are happier
surrounded by other cells and glia.
Good luck,
Matt